Example: stock market

Stability indicating quantitative RP-HPLC method ...

Available online Journal of Chemical and Pharmaceutical Research, 2015, 7(4):346-355. ISSN : 0975-7384. Research Article CODEN(USA) : JCPRC5. Stability indicating quantitative RP-HPLC method development and validation for simultaneous determination of metformin hydrochloride and saxagliptin in bulk and combined tablet dosage form Mohammad Yunoos*1 and D. Gowri Sankar2. 1. Department of Pharmaceutical Analysis, Bapatla College of Pharmacy, Bapatla, Guntur (Dist.), Andhra Pradesh, India 2. College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, India _____. ABSTRACT. A simple and precise Stability indicating RP-HPLC method was developed and validated for the simultaneous determination of Metformin Hydrochloride and Saxagliptin in pure drug and combined tablet dosage form. Chromatography was carried out on Hypersil ODS C18 ( , 5 particle size) analytical column using a mobile phase of Phosphate buffer (KH2PO4) adjusted to pH with dilute orthophosphoric acid, acetonitrile and methanol in the ratio of 25:50:25 % v/v/v at a flow rate of ml/min.

Available online www.jocpr.com Journal of Chemical and Pharmaceutical Research, 2015, 7(4):346-355 Research Article ISSN : 0975-7384 CODEN(USA) : JCPRC5

Tags:

  Stability, Quantitative, Indicating, Stability indicating quantitative

Information

Domain:

Source:

Link to this page:

Please notify us if you found a problem with this document:

Other abuse

Transcription of Stability indicating quantitative RP-HPLC method ...

1 Available online Journal of Chemical and Pharmaceutical Research, 2015, 7(4):346-355. ISSN : 0975-7384. Research Article CODEN(USA) : JCPRC5. Stability indicating quantitative RP-HPLC method development and validation for simultaneous determination of metformin hydrochloride and saxagliptin in bulk and combined tablet dosage form Mohammad Yunoos*1 and D. Gowri Sankar2. 1. Department of Pharmaceutical Analysis, Bapatla College of Pharmacy, Bapatla, Guntur (Dist.), Andhra Pradesh, India 2. College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, India _____. ABSTRACT. A simple and precise Stability indicating RP-HPLC method was developed and validated for the simultaneous determination of Metformin Hydrochloride and Saxagliptin in pure drug and combined tablet dosage form. Chromatography was carried out on Hypersil ODS C18 ( , 5 particle size) analytical column using a mobile phase of Phosphate buffer (KH2PO4) adjusted to pH with dilute orthophosphoric acid, acetonitrile and methanol in the ratio of 25:50:25 % v/v/v at a flow rate of ml/min.

2 The analyte was monitored using PDA. detector at 211 nm. The retention time was found to be min and min for Metformin Hydrochloride and Saxagliptin respectively. Linearity was observed in the concentration range of 125-750 g/ml and g/ml for both Metformin Hydrochloride and Saxagliptin with correlation coefficient of respectively. The mean %. recoveries obtained for Metformin Hydrochloride and Saxagliptin were found to be and respectively. Stress testing which covered acid hydrolysis, base hydrolysis, peroxide, photolytic (UV light), neutral and thermal degradation was performed to prove the specificity of the proposed method and degradation was achieved. The developed method has been statistically validated according to ICH guide lines and found to be simple, precise and accurate with the prescribed values. Thus the proposed RP-HPLC method was successfully applied for the Stability indicating simultaneous estimation of Metformin Hydrochloride and Saxagliptin in routine quality control analysis in bulk and marketed formulations.

3 Keywords: Metformin Hydrochloride, Saxagliptin, RP-HPLC , Forced degradation, method validation. _____. INTRODUCTION. Saxagliptin: Chemically, (as shown in figure 1) it is (1s, 3s, 5s)-2-[(2s)-2-Amino-2-(3-hydroxy-1-adama ntyl) acetyl]-2- azabicyclo [ ] hexane-3-carbonitrile. It has a molecular formula of C18H25N3O2 and molecular weight of g/mol. Saxagliptin is a new oral hypoglycemic agent, dipeptidyl peptidase-4 (DPP-4) inhibitor and antidiabetic for the treatment of type 2 diabetes. DPP-4 inhibitors are a class of compounds that work by affecting the action of natural hormones in the body called incretins. Incretins decrease blood sugar by increasing consumption of sugar by the body, mainly through increasing insulin production in the pancreas, and by reducing production of sugar by the liver. The inhibition of DPP-4 increases levels active of glucagon like peptide 1 (GLP-1), which inhibits glucagon production from pancreatic alpha cells and increases production of insulin from pancreatic beta cells [1-7].

4 346. Mohammad Yunoos and D. Gowri Sankar J. Chem. Pharm. Res., 2015, 7(4):346-355. _____. : Chemical structure of Saxagliptin Metformin Hydrochloride: Chemically, (as shown in ) it is (3-(diamino methylidene)-1, 1-dimethylguanidine; hydrochloride. It has molecular formula of C4H12 ClN5 and molecular weight is g/mol. Metformin is an oral antihyperglycemic agent (type 2 diabetes) belongs to class of biguanides and useful for treating non-insulin-dependent diabetes mellitus (NIDDM). Metformin decreases blood glucose levels by decreasing hepatic glucose production, decreasing intestinal absorption of glucose, and improving insulin sensitivity by increasing peripheral glucose uptake and utilization. These effects are mediated by the initial activation by metformin of AMP-activated protein kinase (AMPK), a liver enzyme that plays an important role in insulin signaling, whole body energy balance, and the metabolism of glucose and fats. Fig. 2: Chemical structure of Metformin Hydrochloride Literature survey reveals that few analytical methods were reported like RP-HPLC methods [8-14], GC method [15], HPTLC methods [16-17] and spectrophotometric methods [18-19] in single or in combination with other drugs in pharmaceutical dosage forms.)

5 However there was no Stability indicating method reported for this drug combination and hence the present study was aimed to develop a simple, fast, economical, selective, accurate, precise and sensitive Stability indicating RP-HPLC method for the simultaneous determination of Metformin Hydrochloride and Saxagliptin in bulk and combined tablet dosage forms suitable for routine quality control analysis. EXPERIMENTAL SECTION. Chemicals: Metformin Hydrochloride and Saxagliptin were obtained as gift samples from 's Laboratories, Hyderabad. HPLC grade water, methanol and acetonitrile were purchased from Chem. ltd., Mumbai. All the chemicals used were of analytical reagent grade ( ). Fixed dose combination tablet formulations (Kombiglyze XR). containing 500 mg of Metformin and 5 mg of Saxagliptin (manufactured by Bristol Myers Squibb India Pvt. Ltd, Mumbai) were procured from local pharmacy market. Instrumentation: quantitative HPLC was performed on Waters technologies 2695 series and PDA detector module equipped with auto injector using empower software.

6 A reverse phase Hypersil ODS C18 (250x , particle size 5 m). analytical column was used. Weighing was done on shimadzu balance (AX 200) and pH adjustments were done using pH meter (ELICO LI 615) was used. Chromatographic conditions: Chromatographic separation and analysis were carried out on Hypersil ODS C18 column ( , 5 particle size) column. The optimized mobile phase consisting of Phosphate buffer (pH adjusted to with dilute orthophosphoric acid), acetonitrile and methanol in the ratio of 25:50:25 %v/v/v. Flow rate was maintained at ml/min and run time for 7 min. Prior to sample injection, column was saturated with mobile phase for 30 min and injection volume of 10 l was injected using auto sampler mode. The detection response was measured at 211 nm at ambient temperature (30 0C). Preparation of Phosphate buffer: Accurately weighed and transferred a quantity of gm of potassium dihydrogen orthophosphate into a 1000 ml of volumetric flask. Added about 900 ml of HPLC grade water and degas to sonicate for 5 min and finally made up the volume with water and then pH of the solution was adjusted to with dilute ortho phosphoric acid.

7 The buffer was subjected to filtration using membrane filter. 347. Mohammad Yunoos and D. Gowri Sankar J. Chem. Pharm. Res., 2015, 7(4):346-355. _____. Preparation of mobile phase: Phosphate buffer (adjusted to pH with dilute OPA), acetonitrile and methanol were taken in the ratio of 25:50:25. %v/v/v and mixed well and then degassed by subjecting to sonication for 10 minutes and filtered under vaccum filtration. Preparation of diluent: Mobile phase was used as diluent. Preparation of Standard solutions: Accurately weighed and transferred 50 mg of Metformin and 5 mg of Saxagliptin reference standards into a 10 ml and 100 ml clean dry volumetric flasks separately and added 3/4th volume of diluent , sonicated for 5 min and made up to the final volume with diluent. From the above stock solution, ml was pipette out in to a 10 ml volumetric flask and then made up to the final volume with diluent to obtained concentration of 500 g/ml of Metformin and 5 g/ml of Saxagliptin working standard solutions respectively.

8 Preparation of sample solutions: 20 tablets were weighed and determined the average weight and then crushed to a fine powder. The tablet powder equivalent to 500 mg of Metformin and 5 mg of Saxagliptin was weighed accurately and transferred into a 100 ml volumetric flask, 70 ml of diluent was added and sonicated for 10 min and then made up the volume with diluent and filtered. From the filtered solution, ml was pipette out into a 10 ml volumetric flask and made up to the mark with diluent to obtained concentration of 500 g/ml of Metformin and 5 g/ml of Saxagliptin working sample solutions. Injected 10 l of filtered portion of the sample and standard preparation into the chromatographic system. Record the responses for the major peaks. Calculated the content of Metformin and Saxagliptin present in each tablet. method validation: System suitability: System suitability test should be carried out to verify that the analytical system is working properly and can give accurate and precise results.

9 Standard solutions were prepared as per the test method and injected five times into the chromatographic system. The system suitability parameters were evaluated for tailing factor, retention times and theoretical plates of standard chromatograms. The results obtained are reported. Accuracy: The accuracy of an analytical method is the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. The study was performed by making three different concentrations at 50%, 100% and 150% levels using standard addition method where sample preparations were spiked with known amount of standard and then each concentration was injected three times into the chromatographic system. The accuracy of an analytical method should be established across its range. System Precision and method precision: The system precision was carried out to ensure that the analytical system is working properly by injecting standard solution preparations six times into the HPLC and the retention time and peak areas were measured and % RSD was calculated.

10 In method precision, a homogenous sample containing of Metformin and Saxagliptin of a single batch were analyzed six times and % RSD was calculated. Specificity: Specificity is the ability to assess unequivocally the analyte in the presence of compounds that may be expected to present, such as impurities, degradation products and matrix components. The specificity of the method was assessed by comparing the Chromatograms obtained from the drug standards with that of obtained from the tablet preparations .The retention times of the drug standards and the drug from sample preparations were same, so the method was specific without interference from excipients in the tablets. Linearity: The linearity of an analytical method was carried out to check its ability to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to the concentration of analyte in samples within a given range. Different concentrations of standard solutions were prepared by diluting aliquots ( ml) of standard stock solution (5000 g/ml of Metformin and 50 g/ml for Saxagliptin) in to 10 ml volumetric flasks to obtained concentrations in the range of 125-750 g/ml for Metformin and g/ml for Saxagliptin and then injected each concentration into the chromatographic system and the chromatograms were recorded.


Related search queries